On the mechanism of isotope exchange kinetics of single protons in bovine pancreatic trypsin inhibitor

Hilton, B.D.; Woodward, C.K.

Biochemistry 18(26): 5834-5841


ISSN/ISBN: 0006-2960
PMID: 42434
DOI: 10.1021/bi00593a013
Accession: 068518768

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The hydrogen isotope exchange kinetics of the slowest exchanging proton resonances in the bovine pancreatic trypsin inhibitor NMR spectrum were measured from pH 1-12, 33-68.degree. C. The pH dependence and the apparent activation energy for each proton vary with temperature. The kinetics for each proton are explained by a model in which exchange is governed by 2 discrete conformational processes that differ in temperature dependence. One process is related to thermal unfolding, and kinetics for exchange by this pathway are of high activation energy, .apprx. 60 kcal/mol, and .apprx. 1/2 order in OH-ion. The second is a dynamical process of the folded conformation, and kinetics for exchange by this process give an activation energy of 20-35 kcal/mol with variable pH dependence approaching 1st order in catalyst ion. Since the chemical exchange step has an activation energy of .simeq. 20 kcal/mol, the enthalpies of the 2 conformational processes are .simeq. 40 and 0-15 kcal/mol, respectively. The model is simple, has a precedent in the hydrogen-exchange literature, and predicts the complex features of the pH and temperature dependence of the single proton exchange rates. For the 2 slowest exchanging protons, the rates at 51.degree. C show a pH-independent plateau between pH 8.4-9.6. In the context of the model, comparison with data for the same resonances at 45.degree. C suggests that the high activation energy conformational process is rate limiting at pH > 8.4, 51.degree. C, and the rate of exposure to solvents is equal to the observed exchange rate, 5 .times. 10-2 h-1.